peak (label) and a sharp beta-sheet feature [(e), [ 1600 cm
-1
], respectively. This
evolution indicated the transition from a beta-sheet to a random-coil motif in the
F23 region, exactly opposite to what was observed for V17. The kinetics for the
evolution of this feature for the F23 sample are shown in (f) and occur on a
timescale of several hours. Importantly, the F23 peak increases and afterwards
decreases, typical for what is expected for an intermediate in the formation process.
This intermediate was observed by isotope labeling throughout the entire FGAIL
region (residues 23–27), what highlights the importance of this beta-sheet type
structure over an extended region.
To test the importance of this region for fibril formation, a macrocycle binding
approach was adopted and the kinetics of the fibril formation upon region-specific
binding to the hIAPP were thoroughly screened. As a result, it was found that only
targeting the FGAIL region with a macrocycle (Mac 21–27 ) slowed down the kinetics
markedly. It was thus inferred that the macrocyclic recognition of the FGAIL region
stabilizes an intermediate on the potential energy curve along fibril formation
(Fig. 7g) with parallel beta-sheet arrangement (parallel red arrows). Overall, this
extensive study of fibril formation nicely demonstrated the value of 2D IR
spectroscopy in real time and in combination with isotope labelling for the
elucidation of intermediates in the process of protein misfolding.
Similar examples exist that have supported the value of 2D IR spectroscopy
regarding structural biology. Zanni’s group has continuously refined and expanded
the model of fibril formation in hIAPP over several years, which eventually led to
the conclusive picture presented above [91–93]. However, labelling of a single
residue limits this approach to rather small proteins due the spectral resolution of the
label in the congested spectra, and additionally requires the preparation of a large
amount of labelled variants, as demonstrated. Other approaches have also been
shown to allow the investigation of much larger proteins. Specific regions from the
amino acid sequences in proteins as large as 173 residues have been resolved by use
of segmental isotope-labelling [94]. This approach on the one hand significantly
increases the number of absorbing isotope-substituted residues and is equally
applicable to smaller proteins [81], but on the other hand might be limited to
proteins, which can be expressed in different parts.
In other studies, the same group recently also used 2D IR to investigate in detail
ion configurations in the selectivity filter of a potassium ion-channel [95]. The
authors exploited 2D IR in combination with MD simulations to reveal ion
distributions in the channel and to compare the results with proposed mechanisms
for ion-permeation. The investigations were made possible via an experimental
approach termed ‘‘semi-synthesis’’, in which the particular protein under study is
assembled from different parts, i.e. a synthetic peptide that can be modified on
demand, and other recombinant peptides that constitute the remaining protein parts.
Such an approach therefore significantly increases the synthetic flexibility for
sample preparation. Together with the possibility to incorporate unnatural amino
acids [96, 97]. with tailored IR-labels, these methods constitute now a significantly
broadened applicability of 2D IR for the corresponding structural investigations of
proteins.
Top Curr Chem (Z) (2017) 375:86
123
133
Reprinted from the journal
-1
], respectively. This
evolution indicated the transition from a beta-sheet to a random-coil motif in the
F23 region, exactly opposite to what was observed for V17. The kinetics for the
evolution of this feature for the F23 sample are shown in (f) and occur on a
timescale of several hours. Importantly, the F23 peak increases and afterwards
decreases, typical for what is expected for an intermediate in the formation process.
This intermediate was observed by isotope labeling throughout the entire FGAIL
region (residues 23–27), what highlights the importance of this beta-sheet type
structure over an extended region.
To test the importance of this region for fibril formation, a macrocycle binding
approach was adopted and the kinetics of the fibril formation upon region-specific
binding to the hIAPP were thoroughly screened. As a result, it was found that only
targeting the FGAIL region with a macrocycle (Mac 21–27 ) slowed down the kinetics
markedly. It was thus inferred that the macrocyclic recognition of the FGAIL region
stabilizes an intermediate on the potential energy curve along fibril formation
(Fig. 7g) with parallel beta-sheet arrangement (parallel red arrows). Overall, this
extensive study of fibril formation nicely demonstrated the value of 2D IR
spectroscopy in real time and in combination with isotope labelling for the
elucidation of intermediates in the process of protein misfolding.
Similar examples exist that have supported the value of 2D IR spectroscopy
regarding structural biology. Zanni’s group has continuously refined and expanded
the model of fibril formation in hIAPP over several years, which eventually led to
the conclusive picture presented above [91–93]. However, labelling of a single
residue limits this approach to rather small proteins due the spectral resolution of the
label in the congested spectra, and additionally requires the preparation of a large
amount of labelled variants, as demonstrated. Other approaches have also been
shown to allow the investigation of much larger proteins. Specific regions from the
amino acid sequences in proteins as large as 173 residues have been resolved by use
of segmental isotope-labelling [94]. This approach on the one hand significantly
increases the number of absorbing isotope-substituted residues and is equally
applicable to smaller proteins [81], but on the other hand might be limited to
proteins, which can be expressed in different parts.
In other studies, the same group recently also used 2D IR to investigate in detail
ion configurations in the selectivity filter of a potassium ion-channel [95]. The
authors exploited 2D IR in combination with MD simulations to reveal ion
distributions in the channel and to compare the results with proposed mechanisms
for ion-permeation. The investigations were made possible via an experimental
approach termed ‘‘semi-synthesis’’, in which the particular protein under study is
assembled from different parts, i.e. a synthetic peptide that can be modified on
demand, and other recombinant peptides that constitute the remaining protein parts.
Such an approach therefore significantly increases the synthetic flexibility for
sample preparation. Together with the possibility to incorporate unnatural amino
acids [96, 97]. with tailored IR-labels, these methods constitute now a significantly
broadened applicability of 2D IR for the corresponding structural investigations of
proteins.
Top Curr Chem (Z) (2017) 375:86
123
133
Reprinted from the journal